Autofluorescence Dynamics of Anaerobic Bacterial Strains Under 405 nm Laser Excitation–Quantitative Hue-Based Analysis

Autofluorescence induced by 405 nm excitation may provide a non-invasive optical approach for characterizing cultured bacteria. This study evaluated the dynamics of autofluorescence in anaerobic bacterial strains using quantitative hue analysis. Twelve reference strains representing 11 bacterial species were cultured under controlled anaerobic conditions. Eight independent colonies per strain were evaluated repeatedly at 24, 48, 72, 96, and 168 h after inoculation, using 405 nm laser excitation for 45 s at each time point. Autofluorescence color was assessed using ΔH, defined as the mean colony hue minus the agar background hue. The effects of bacterial strain, incubation time, and their interaction were analyzed using ART-ANOVA. Significant effects were observed for both factors and their interaction (p < 0.001). Comparatively higher, less negative ΔH values were observed for A. odontolyticus, B. fragilis, B. breve, C. acnes, and C. sporogenes ATCC 2486, whereas more negative values were observed for P. intermedia, P. micra, and A. israelii. P. intermedia showed the largest visible shift in mean ΔH, particularly between 48 and 72 h. Temporal changes were not consistently monotonic, and several strains showed partial reversals at later time points. These findings indicate strain-dependent temporal changes in colony color relative to the culture medium. Absolute fluorescence intensity was not quantified. Quantitative hue analysis may provide complementary information for characterizing cultured anaerobic bacteria, but its reproducibility and performance for identifying unknown isolates require further validation.